Cytokeration (5/18) Antibody Staining Protocol for Immunohistochemistry


Description: NCL-C50 reacts with human cytokeratin intermediate filament proteins of 58kD and 45kD, identified as cytokeratins 5 and 18, respectively. Cytokeratin 5 is expressed by basal cells and suprabasal cells of stratified epithelium. Cytokeratin 18 is expressed by simple epithelial cells, including ductal and glandular epithelia.


Primary Antibody

Name: Cytokeratin 5/18 Antibody

Clone: C-50, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-C50

Dilution: 1:50 - 1:100 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 minutes/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Cytoplasmic
Images: Search image

Additional Information:
Tissue Type: Skin
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections.
Positive Control: Skin
Negative Control: Omit primary antibody, isotype control or absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary


There is no need to perform antigen retrieval for frozen sections.